Bioluminescence Imaging of Acinetobacter baumannii-Infected Burn Wounds in Mice

Samenvatting

Source: Wang, Y., et al. In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging. J. Vis. Exp. (2017)

This video demonstrates the procedure for imaging burn wound infections in mice inoculated with Acinetobacter baumannii carrying a bioluminescent operon. It demonstrates how luciferase-catalyzed substrate oxidation produces light proportional to bacterial load, enabling real-time monitoring of infection progression using a bioluminescence imaging system.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Bioluminescence Imaging of Infections in Mice

1. Image the mice using a low-light imaging system that includes an intensified charge-coupled device camera, a camera controller, a specimen chamber, and an image processor.
2. Anesthetize the mice with an intraperitoneal injection of a ketamine-xylazine cocktail (100 mg/kg - 20 mg/kg). Lightly touch the palpebral of the mice with a cotton swab; an absence of the palpebral reflex suggests an appropriate anesthetic depth.
3. Start the live imaging software. In the control panel that appears, click Initialize. Wait until the color of the Temperature box turns green, indicating that the temperature of the stage in the specimen chamber has reached 37 °C.
4. Place the mice on the stage (37 °C) in the specimen chamber of the imaging system, with the infected burns directly under the camera.
   NOTE: The bioluminescence of the bacteria could decrease when the burns become dry. Therefore, it is recommended to moisturize the mouse burns with PBS before imaging.
5. In the control panel, put a check mark next to "Luminescence." Select "Auto exposure" so that the exposure time for imaging will be optimized by the live imaging software based on the bioluminescence intensity.
6. Select "C" from the "Field of View" drop-down list. Select the "Scan mid range" option to let the software determine the focal distance. Put a check mark next to Overlay.
7. Click "Acquire" to capture the image. In the "Edit Image Label" box, click "OK;" an "Image Window" and "Toll Palette" will appear.
8. Set Auto ROI parameters for auto-selection.
9. Quantify the bioluminescence intensity as relative luminescence units (RLUs) and display the bioluminescence in a false-color scale ranging from pink (most intense) to blue (least intense).
10. Calculate the survival fraction of the bacteria in mouse burns at varying time points based upon bioluminescence intensity analysis. The survival fraction of bacteria at a given time point = the bioluminescence intensity measured at that time point / the bioluminescence intensity measured right before antimicrobial blue light exposure.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
IVIS	PerkinElmer Inc, Waltham, MA	IVIS Lumina Series III	Pre-clinical in vivo imaging
Light-emitting diode LED	VieLight Inc, Toronto, Canada	415 nm	Light source for illumination
Mouse	Charles River Laboratories, Wilmington, MA	BALB/c	7-8 weeks age, 17-19 g weight
Acinetobacter baumannii	Brooke Army Medical Center, Fort Sam Houston, TX	Clinical isolate	Engineered luminescent strain
Greiner dishes	Sigma-Aldrich Co. LLC	P5112-740EA	35 mm ×10 mm